Purification And Action Mechanism Of ACE Inhibitory Peptides From Wakame(Undaria Pinnatifida)

Hypertension is a major risk factor responsible for the most common cardiovascular diseases and diabetes.Inhibiting the ACE activity is considered as a useful treatment against hypertension and ACE inhibitors are primary drugs to treat hypertension.However,synthetic angiotensin-I-converting enzyme(ACE)inhibitors,such as lisinopril,captopril and enalapril have some adverse side-effects,which include dry cough,abnormal taste buds,anaphylaxis.Therefore,the safety of ACE inhibitory peptides extracted from food,especially from marine organisms,has become a research hotspot.However,there are still some problems in the research of ACE inhibitory peptides,such as low content of ACE inhibitory peptides in complex macromolecular components,complex separation and purification process,and less research on the interaction between ACE inhibitory peptides and ACE.In this thesis,the wakame was taken as the research object.Based on the idea of denatured protein,immobilized enzyme and biological affinity purification,wakame protein was denatured by ultrasound.The ACE derived from pig lung was immobilized onto magnetic metal organic frameworks(MOF)and a novel immobilization system was constructed.A new ACE inhibitory peptide was obtained by affinity purification using Fe₃O₄@ZIF-90-ACE.Finally,the mechanism of ACE inhibitory peptide KNFL derived from wakame was explored.The major results were as follows:(1)The ACE inhibitory peptides were separated from wakame by ultrafiltration,gel filtration column chromatography and reversed high performance liquid chromatography(RP-HPLC).The most active components were identified as Tyr-Lys-Tyr-Tyr(YKYY,IC₅₀=71.88±1.43?M)by a mass spectrometer which was retrieved as reported inhibitory peptides.Less purification steps was used in this experiment and bromelain used for enzymatic hydrolysis was more safe and economical.(2)The optimum conditions were observed at ultrasonic power of 200 W,treatment time of 15 min and intermittent ratio 1:2(s/s).Under the optimal ultrasonic conditions,the ACE-inhibition activity was increased from 41.20±2.0%to 88.10±2.1%,the degree of hydrolysis(DH)was enhanced from 9.20±0.2%to15.60±0.3%and the content of peptide was increased from13.61±0.2%to19.50±0.2%.The results showed that ultrasonic pretreatment could promote the enzymatic hydrolysis of wakame protein to higher ACE inhibition.The results of spectroscopy indicated that the secondary structure of protein was changed by ultrasonic pretreatment,which was mainly reflected in the decrease of?-helix and?-turn content and the increase of?-sheet and random coil content.With the looseness of protein structure,active peptides were more easily released in the process of enzymatic hydrolysis,and more hydrophobic amino acids were exposed to the outside and produced by hydrolysis.In addition,the solubility,emulsification activity,surface hydrophobicity of the protein were also improved by ultrasonic pretreatment.(3)The ACE crude extract from pig lungs was immobilized onto Fe₃O₄@ZIF-90 by adsorption-crosslink.The optimum immobilization conditions were as follow:the protein concentration of 10.0 mg·m L^-1,immobilization pH of8.0,immobilization temperature of 45?and immobilization time of 2.0 h.Under the optimal conditions,the enzyme activity of Fe₃O₄@ZIF-90-ACE was0.028±0.006 U·g^-1.The results showed that the optimum pH of immobilized enzyme was 8.3 and the optimum temperature was 45?.Compared with the free ACE,immobilized ACE was less sensitive to temperature and the optimal temperature of the enzyme was increased.Stability results of free and immobilized ACE suggested that immobilized ACE had better thermal,pH and storage stability than the free ACE.The relative activity decreased with the increase of the number of cycles,but the activity retention rate of immobilized ACE was 54.05%after the sixth cycle.The mechanism of separation and immobilization of ACE on Fe₃O₄@ZIF-90 were investigated from the aspects of binding energy,metal ion affinity chromatography and bonding by density function theory(DFT)and X-ray photoelectron spectroscopy(XPS).The results showed that Zn²⁺could coordinate with imidazolyl(histidine),thiol sulfur(tryptophan),and indolyl(cysteine)functional groups in ACE.ACE was not only physically adsorbed on the support,but also bonded to the Fe₃O₄@ZIF-90skeleton chemically.(4)The active peptide with the highest ACE inhibitory activity was extracted from Wakame protein by magnetic affinity chromatography(Fe₃O₄@ZIF-90-ACE)combined with RP-HPLC.The amino acid sequence of the peptide was identified as Lys-Asn-Phe-Leu(KNFL,IC₅₀=225.87±2.7?m)by MALDI-TOF-TOF MS/MS which was retrieved as a novel inhibitory peptides.Compared with conventional separation technologies,such as ion exchange chromatography and gel filtration chromatography,affinity purification for the preparation of ACE inhibitory peptides has attracted widespread attention due to its simple process and less time.Moreover,the preparation of Fe₃O₄@ZIF-90-ACE was easily obtained by shaking without additional activation.In addition,immobilization of crude ACE could not only save cost,but also improve stability and operability.(5)The interaction between ACE inhibitory peptide KNFL and ACE was investigated by initial concentration method,isothermal titration calorimetry(ITC),multiple spectroscopic techniques and molecular dynamics simulations,and the inhibition model was established.The inhibitory type of inhibitory peptide KNFL on ACE was mixed-type.The binding of KNFL to ACE was a spontaneous exothermic process driven by enthalpy and entropy.KNFL had multiple binding sites on ACE,the hydrogen bonds of KNFL interacting with the active and non-active site of ACE were generated,and the molecular diameter of ACE was increased.The ACE inhibitory peptide(KNFL)had fluorescence quenching effect on ACE and the microenvironment surrounding Trp-and Tyr-residues in ACE was affected.The circular dichroism(CD)revealed that the content of?-helix and random coil in ACE were decreased and the content of?-folded was increased,which caused the rearrangement of the peptide chain and the change of ACE structure,and then the activity of ACE was decreased.The stability of complexes(ACE-KNFL)was investigated by molecular dynamics(MD)simulation.The results of root mean square deviation(RMSD),root mean square fluctuation(RMSF)and radius of gyration(Rg)showed that the docking complex(ACE-KNFL)was stable under the simulated conditions.Finally,the inhibition model was established based on free energy binding scenarios theory.The complex could be formed by the multiple steps of inducing fit and conformational selection.